Charge-Conversion Strategies for Nucleic Acid Delivery

Abstract

Nucleic acids are now considered as one of the most potent therapeutic modalities, as their roles go beyond storing genetic information and chemical energy or as signal transducer. Attenuation or expression of desired genes through nucleic acids have profound implications in gene therapy, gene editing and even in vaccine development for immunomodulation. Although nucleic acid therapeutics bring in overwhelming possibilities towards the development of molecular medicines, there are significant loopholes in designing and effective translation of these drugs into the clinic. One of the major pitfalls lies in the traditional design concepts for nucleic acid drug carriers, viz. cationic charge induced cytotoxicity in delivery pathway. Targeting this bottleneck, several pioneering research efforts have been devoted to design innovative carriers through charge-conversion approaches, whereby built-in functionalities convert from cationic to neutral or anionic, or even from anionic to cationic enabling the carrier to overcome several critical barriers for therapeutics delivery, such as serum deactivation, instability in circulation, low transfection and poor endosomal escape. This review will critically analyze various molecular designs of charge-converting nanocarriers in a classified approach for the successful delivery of nucleic acids. Accompanied by the narrative on recent clinical nucleic acid candidates, the review concludes with a discussion on the pitfalls and scope of these interesting approaches.

Keywords: Charge Conversion; Drug Delivery; Endosomal Escape; Non-cationic; Nucleic Acid.

Figure 1.

Schematic diagram of various classified charge-conversion approaches for nucleic acid delivery

Figure 2.

(a) Non-cationic polymer-RNA complex formation in aqueous media. The decationized complex was prepared via a crosslinking reaction that removed the positive charge (reproduced with permission from reference , Copyright 2019 American Chemical Society); (b) Tri-component self-assembly of a cationic polymer, an anionic siRNA and zwitterionic lipids via solvophobic forces and physical incarcerations with crosslinking to form L-siP nanoassembly; (c) left: Lipid-coated L-siP nanoassembly constructed with CG–MD simulation, green-polymer, yellow-oligonucleotide, transparent gray-lipid later; right: N-STORM confocal microscopy image of an individual L-siP nanoassembly, red- cy3-siRNA, green- carboxyfluorescein-labeled DSPE-PEG lipid, scale: 100 nm; (d) Silencing of eGFP gene in HeLa cells with L-siP nanoassembly showing reduction of green fluorescence, scale bar: 20 μm; (e) Cytotoxicity measurement via cell viability in HeLa cells with Lipofectamine-negative control siRNA complex (LF-siNC), empty L-siP and L-siP loaded with negative control siRNA (L-siP/siNC); (f) Cell membrane damage assessment with LDH-assay in HeLa cells showing no apparent membrane damage upon treatment with L-siP nanoparticles; (g) Uptake of doxorubicin increased in L-siP/siMDR1 treated NCI-ADR/RES cells, resistant to doxorubicin, scale: 10 μm (reproduced with permission from reference , Copyright 2019 American Chemical Society).

Figure 3.

(a) Concept of decationized polyplexes for improved biodistribution and safety profile; (b) pDNA encapsulation in crosslinked pHDP-PEG polyplexes that can be decationized via hydrolysis reaction; (c) Safety evaluation by cell viability measurement in HUVEC cells upon treatment with non-cationic pHDP-PEG polyplex, cationic control pHP-PEG and polyethylene imine (PEI) where decationized polyplex shows better safety profile compared to cationic controls; (d) Images of zebrafish embryos upon treatment with increasing dosages of decationized pHP-PEG and its cationic control revealing elevated levels of toxicity only for cationic control and PEI in comparison to decationized pHP-PEG polyplex, * = significant mortality, ** = significant developmental defect (reproduced with permission from reference , Copyright 2014 Elsevier).

Figure 4.

(a) mRNA delivery strategy with CART systems, incorporated with charge-altering mechanism, to induce intracellular protein expression; (b) Proposed cationic charge-alteration mechanism for oligo(α-amino ester)s involving two sequential 5- and 6-membered transition states; (c) MTT cell viability assay over 72 h in HeLa cells showing low toxicity of CART/mRNA complexes; (d) eGFP mRNA transfection efficacy in different cell lines with a preferred CART system (denoted as 7: with 13 lipid and 11 cationic chains, where n=13, m=11, R=3) (reproduced with permission from reference , Copyright 2017 The National Academy of Sciences of the USA).

Figure 5.

(a) CART-pPKCδ-GFP transfected CHO-K1 cells expressing pPKCδ-GFP fused protein in cytosol. The fused protein was able to translocate to cell membrane upon treatment with bryostatin 1, attesting the structure and activity of expressed fused protein (reproduced with permission from reference , Copyright 2018 American Chemical Society); (b) Fluorescently labeled single- and mixed-lipid CART systems; (c) Bioluminescence studies for the in vivo delivery of luciferase mRNA in BALB/c mice with mixed lipid CART system (CART 13, shown in Figure 5b) showing high levels of gene expression in spleen, considered as a favorable indication of lymphocyte transfection; (d) Transfection of immune cells for single- vs. mixed-lipid CART systems in spleen (reproduced with permission from reference , Copyright 2018 The National Academy of Sciences of the USA); (e) Proposed charge-altering rearrangement mechanism for poly(serine ester) CART system (reproduced with permission from reference , Copyright 2019 American Chemical Society).

Figure 6.

(a) ROS-responsive cationic-to-anionic charge conversion for B-PDEAEA polymer to generate anionic polyacrylic acid; (b) Complexation of pDNA with B-PDEAEA polymer and intracellular gene delivery: 1. complexation, 2. targeting lipid coating, 3. membrane fusion, 4. ROS-responsive release of DNA, 5. localization of DNA in nucleus (reproduced with permission from reference , Copyright 2016 Wiley-VCH Verlag GmbH & Co); (c) Structural modifications to introduce esterase-responsiveness in PQDEA polymer and the proposed charge-reversal mechanism (reproduced with permission from reference , Copyright 2018 American Chemical Society).

Figure 7.

(a) Chemical structure of the BMAAD polymer and Cryo-TEM images of the formed micelles with charge-conversion at different pH (reproduced with permission from reference , Copyright 2013 American Chemical Society); (b) Synthesis of PAD polymer and charge-shifting mechanism through hydrolysis that facilitated the release of nucleic acid (reproduced with permission from reference , Copyright 2020 American Chemical Society); (c) Propionic 4-acetoxybenzyl ester modified PEI reversed cationic charge via esterase-mediated hydrolysis to form a zwitterionic species (reproduced with permission from reference , Copyright 2016 Wiley-VCH Verlag GmbH & Co).

Figure 8.

(a) Supramolecular assembly between DNA and multi-walled vesicles of a charge-reversing lipid amphiphile, and the release mechanism of nucleic acid via hydrolysis of ester moieties present in the lipid to form anionic species; (b) Structures of charge-reversing (1) and control lipids (2–5), wherein the favored prototype 1 was equipped with quaternary ammonium headgroup (for nucleic acid complexation), hydrophobic acyl chain (for bilayer formation) and terminal benzyl ester (for hydrolysis driven anionic group generation); (c) Kinetic measurements of fluorescence from ethidium bromide displacement assay to show the release of DNA from the assembly in response to porcine liver esterase (reproduced with permission from reference , Copyright 2004 American Chemical Society); (d) A supramolecular assembly of cation lipid with tripeptide headgroup and anionic nucleic acid, and light-triggered disassembly to release nucleic acids via charge conversion to generate anionic carboxylate groups; (e) Zeta potential studies showing charge-reversal of different tripeptide lipid-pDNA assemblies upon exposure with UV light (reproduced with permission from reference , Copyright 2016 Elsevier).

Figure 9.

(a) Schematic for charge-reversing PIC micelle strategy for pDNA delivery, wherein the anionic ternary polyplex reverses to cationic under acidic pH condition in endosome causing endosomal disruption; (b) Structure of cationic pAsp(DET), charge-reversing pAsp(DET-Aco), and non-charge reversing control pAsp(EDA-Suc) polymers; (c) Zeta potential measurements to observe charge conversion in pDNA/pAsp(DET)/pAsp(DET-Aco), solid sphere: at pH 7.4, dark sphere: at pH 5.5 (reproduced with permission from reference , Copyright 2008 Wiley-VCH Verlag GmbH & Co.); (d) Utilization of phenylboronic acid-diol interaction for siRNA encapsulation in PIC micelles and its ATP-responsive release (reproduced with permission from reference , Copyright 2012 Wiley-VCH Verlag GmbH & Co.).

Figure 10.

(a) Modular liposome assembled with pH sensitive charge converting complex for siRNA delivery, wherein anionic complex reverses to cationic under acidic tumor environment to enhance uptake and then nucleic acid is released to cytosol via a endosome membrane fusion mechanism (reproduced with permission from reference , Copyright 2018 Royal Society of Chemistry); (b) DNA-g-PCL nanogel formation to deliver CRISPR/Cas9 for intracellular gene editing (reproduced with permission from reference , Copyright 2019 Royal Society of Chemistry).

Figure 11.

(a) Confocal microscopy of Cy5-mRNA treated HeLa cells stained with (TRITC)-Dextran4400 endosomal marker showing escape for D13:A11 CART system and colocalization for non-self-immolative control D13:G12 system, scale: 10 μm (reproduced with permission from reference , Copyright 2017 The National Academy of Sciences of the USA); (b) HUVEC cells transfected with Cy5-pDNA through pAsp(DET) and pAsp(EDA-Suc) polyplexes showing efficient endosomal escape after 24 h for only pAsp(DET) polyplexes, endosome/lysosomes were stained with Lysotracker green, scale: 20 μm (reproduced with permission from reference , Copyright 2008 Wiley-VCH Verlag GmbH & Co.); (c) top: Endosomal colocalization (after 4 h) and escape (after 24 h) of cy3-siRNA delivered via L-siP nanoassembly, endosome stained with Lysotracker blue (pseudo-colored as green), scale: 20 μm; bottom: Calcein assay showing release of calcein dye from endosomes to cytosol only in presence of L-siP nanoassembly, while calcein remain entrapped for control cells, scale: 10 μm (reproduced with permission from reference , Copyright 2019 American Chemical Society).